Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
  • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
  • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof

Definitions

• the present invention relates to new anti-tumor drugs developed from polysaccharidic compounds endowed with the ability to deliver molecules specifically. State of the art
• BA Butyric Acid
• BA is one of the main short-chain fatty acids derived from the colonic fermentation of complex carbohydrates introduced with diet (Hill MJ. Eur J Cancer Prevention 4:897-904, 1995; Cummings JH., Gut 22:763-779, 1981) and is physiologically present in millimolar concentrations in the colon, where it regulates the turnover of colonic epithelial cells by inducing cell differentiation and programmed cell death or apoptosis (Jass JR.
• BA is capable of inhibiting tumor growth according to experimental evidences obtained from a significant series of cell lines representative of the most common human solid tumors (Coradini D, Biffi A, Costa A et al., Cell Prolif 30:149-159,
• HDAC histone de-acetylase enzymes
• Retinoic acid (hereafter referred to as RA) is a monocarboxyilic polyunsaturated acid composed of 20 carbon atoms and represents the oxidized form of vitamin A, well known for its important biological functions in the process of vision, in maintenance of the cutaneous function, in haemopoiesis and in embryonic development (Lotan R, Biochem Biophys Acta 605:33-37,1980; Dower D, Koeffler HP., Exp Cell Res 138:193-199,1982).
• the mechanism of action of RA is strictly correlated with the presence of specific endocellular receptors (retinoic acid receptor, RAR), belonging to the class of ligand-dependent transcription factors, which regulate expression of genes responsible for cell differentiation.
• RAR retinoic acid receptor
• RA and its derivatives have long been used in the treatment of several diseases characterized by abnormal cell proliferation, among which neoplasia (Hansen LA, Sigman CC, Andreola F, et al., Carcinogenesis 21:1271-279,2000; DelUca LM, Kosa K, Andreola F., J Nutr Biochem 8:426- 437,1997).
• hyaluronic acid (hereafter referred to as HA) turned out to be of special practical interest for selective delivery of biologically active molecules whose clinical use is limited by physico-chemical problems.
• hyaluronic acid is recognized by a specific membrane receptor, CD44, that is over-expressed in actively growing cells and particularly in tumor cells (Rudzki Z and Jothy S., J lin Pathol:Mol Pathol 50:57-71 , 1997).
• HA is a polysaccharide consisting of disaccharide units of glucuronic acid and N-acetylglucosamine, with molecular weight up to millions of Daltons, and with partially esterifiable hydroxyl groups.
• the present invention fulfils at the same time the needs for effectively carrying retinoic and butyric acids on one molecule and for solving problems inherent to the simultaneous presence of two active principles on the same carrier molecule.
• Summary of the invention The present invention describes new mixed esters of hyaluronic acid with retinoic and butyric acids. These esters are characterized by a ratio between the degree of substitution with butyric acid and retinoic acid that is at least 6 and more preferably higher than 10.
• a degree of substitution with butyric acid that is preferably ranging from 0.05 to 1.0, even more preferably ranging from 0.1 to 0.35, and by a degree of substitution with retinoic acid ranging from 0.002 to 0.1 , even more preferably ranging from 0.01 to 0.05.
• esters with these characteristics allow to reach a pharmacologically effective concentration of butyric and retinoic acids at the target site, potentiating their biological activity. Indeed, cytostatic and differentiating effects of the two active principles turned out to be even qualitatively and quantitatively greater than those obtained either by individual administration of the two acids or as association of the two mono-esters obtained by esterification with hyaluronic acid.
• the cytostatic and differentiating activities of the above-said esters make possible to treat with greater effectiveness, as compared to current therapies, pathologies characterized by conditions of cell hyper-proliferation, including human solid and systemic tumors.
• the esters of the invention give rise to solutions characterized by low viscosity and good solubility and therefore suitable for pharmaceutical compositions that can be easily administered to humans and animals.
• the present invention includes a process for preparation of the aforesaid esters consisting in the formation of an alcoholate of hyaluronic acid under particular reaction conditions: the alcoholate is first reacted with retinoic acid and then with butyric acid.
• the alcoholate is first reacted with retinoic acid and then with butyric acid.
• Such procedure makes possible to easily obtain the desired degrees of substitution together with a controlled degradation of the native hyaluronic acid molecule within a medium-low molecular weight range.
• Description of the figures Figure 1 Comparison between the anti-proliferative effect of scalar doses of retinoic-butyric mixed esters of hyaluronic acid and of butyric or retinoic monoesters in human mammary carcinoma cells (MCF7).
• the effect on cell growth was assessed by a colorimetric method (MTT) as percentage of inhibition compared to the control, represented by cells maintained in culture medium alone.
• MTT colorimetric method
• Butyric monoester (HA-BA): - ⁇ -; retinoic monoester (HA-RA): -•-; mixed ester of hyaluronic acid with retinoic acid and butyric acids (HA-BA/RA): -A-.
• Figure 6 Effect of retinoate-butyrate mixed ester and of the two active principles (butyric acid and retinoic acid) in the promyelo/monocytic cell line U937.
• the effect on cell cycle and on cell proliferation has been assessed.
• the assessment has been performed by flow cytometry after 3 days of treatment with the mixed ester of the invention at 0.1 mg/ml concentration, or with butyric acid or retinoic acid at concentrations of 0.2 mM and 10 "5 M, respectively.
• Cell cycle perturbations induced by these compounds are related to the effect on cell proliferation (ordinate axis) exerted by identical concentrations of the drugs and expressed as percentage of the control (CTR) represented by cells cultured in medium alone.
• CTR percentage of the control
• Cytometric analysis indicates that the mixed ester induces maximal inhibition already after 3 days of treatment, with 72% of the cells blocked in the Go / i phase of cycle. Instead BA at 0.2 mM concentration is totally unable to induce an anti-proliferative effect or a block of the cell cycle, and 10 "5 M RA significantly inhibits cell growth with a concomitant block in the G 0 / ⁇ phase of the cell cycle.
• CTR control.
• the graph synthetically shows the effects on cell growth (expressed as percentage of cells blocked in the G0 /1 phase of the cycle, % ordinate axis) and effects on differentiation (measured as percentage of cells expressing the CD11b antigen) induced by the various compounds administered individually or in association.
• the figure shows the assessment performed by flow cytometry after 3 days of treatment with the various compounds administered at a concentration corresponding to that in the mixed ester at a dose of 0.1 mg/ml.
• Figure 8 Comparison between the anti-proliferative effect of scalar doses of retinoic-butyric mixed esters of hyaluronic acid and of butyric or retinoic monoesters in the colon adenocarcinoma cell line HT29.
• Butyric mono-ester -A-; Sodium butyrate (NaB) - • -; mixed ester of hyaluronic acid with butyric and retinoic acid (HBR): - ⁇ -.
• the invention describes new mixed esters of hyaluronic acid (HA) with retinoic and butyric acids, also named HBR for the purposes of the present invention.
• HA hyaluronic acid
• butyric acids also named HBR for the purposes of the present invention.
• the hydroxyl groups of hyaluronic acid are partially substituted with the acyl residues of retinoic acid (RA) and butyric acid (BA).
• Said mixed esters are characterized by a higher content of butyric acid than retinoic acid according to a ratio between the degree of substitution (D.S.) with butyrate and with retynoate of at least 6 (DS BA/RA ⁇ 6) or, more preferably, higher than 10.
• said esters have a degree of substitution with butyric acid ranging from 0.05 to 1.0, or even more preferably ranging from 0.1 to 0.35 and a degree of substitution with retinoic acid ranging from 0.002 to 0.1 , or even more preferably ranging from 0.01 to 0.05.
• molecular weight is preferably comprised within the range from 10.000 to 30.000 Daltons.
• molecular weight indicates the average molecular weight (MW) of hyaluronic acid only, without considering the contribution of butyric and retinoic residues.
• degree of substitution designates the number of esterified hydroxyl groups for each repetitive unit of hyaluronic acid (consisting of the dimer GlcNAc- GlcUA).
• retinoic acid or "(RA)” designates all isomeric forms of this compound, hence both its natural form (with all the double bonds in frans-form), and all the other possible isomeric forms.
• the mixed esters of HA described in the present invention simultaneously deliver pharmacologically effective amounts of retinoic and butyric acids to a specific target site of the cell. Therefore both pro-differentiating and cytostatic activities are qualitatively and quantitatively higher than those obtainable with the two acids administered individually or as association of mono-esters with hyaluronic acid.
• the association of the two mono-esters (HA-BA and HA-RA comparatively used) although producing the same effect on growth inhibition is not able to induce any significant effect on cell differentiation. This argues in favour of the remarkable potential of the mixed esters as compared to the active principles and to their mono-esters used separately or in association.
• the anti-proliferative activity of the aforesaid mixed esters is exerted by a cytostatic action that is detectable as cell growth inhibition due to a block of cells in the Goi phase of cell cycle, followed by activation of cell differentiation or, at high concentrations, by ceil death or apoptosis.
• the induction of cell differentiation can be monitored through the re-expression of membrane antigens specific for the tissue under consideration such as the activation of CD11a and CD11b surface antigens in pro-myelocytic cells.
• the mixed esters of the present invention can block tumor growth and restore the process of cell maturation, or induce cell death in a dose-dependent manner in most tumors, thus exerting effects on both the control of disease and on its therapy.
• This makes their application useful for the treatment of both solid and systemic tumors, and can be particularly useful in the therapy of tumors of the promyelocytic lineage and specifically in the acute promyelocytic leukemia (APL), due to their strong pro-differentiating effect.
• APL acute promyelocytic leukemia
• it can be in fact hypothesized a direct mechanism dependent on the presence of the chimeric receptor characteristic of this disease.
• the particularly high biological activity of the esters of the invention can be attributed to their action at two levels: inhibition of HDAC activity (histon-deacetylases that inhibit normal differentiation) through the butyric residues and, at the same time, induction of differentiation through the retinoic residues present on the carrier molecule.
• HDAC activity histon-deacetylases that inhibit normal differentiation
• the esters of the invention induce the re-expression of surface antigens CD11a and CD11b in cells of the pro-myelocytic lineage.
• the presence of the specific hyaluronic acid receptor CD44 on tumor cells is instrumental for the mixed esters to exert these effects.
• the presence of this receptor has been largely shown in most human solid tumors including, for instance, breast, colon and lung adenocarcinomas and melanoma, as well as in some systemic tumors such as APL. Due to their peculiar features, the mixed esters of the invention allow: (i) to achieve a suitable dosage and high bio-availability of both acids at the same site of action, (ii) to maintain the property of site-specificity of hyaluronic acid, used as carrier molecule, (iii) to achieve new pharmacological effects to improve the treatment of tumor pathologies, (iv) to obtain a drug with physico-chemical features that make it easy to administrate, (v) to reduce the toxicity of effective doses of retinoic acid. .
• the activity of said compounds is not simply explained by the sum of the activities of the two active principles (RA and BA), but is rather explained by an unexpected qualitative and quantitative synergism. This is probably due to the simultaneous presence of the two active principles on the same carrier molecule, thus enabling a cellular interaction between RA and BA, unlike the individual esters supplied as mixture.
• the mixed esters of the invention potentiate the effect that is obtained with the simple association of retinoic and butyric acid or their mono-esters with hyaluronic acid.
• esters of the present invention on growth and differentiation of cells expressing the hyaluronic acid receptor (CD44) are totally unexpected, both in terms of behaviour of retinoic and butyric acids, separately or in physical mixture, and of simultaneous administration of single mono-esters of hyaluronic acid.
• the cytometric analysis shows that the greatest inhibition of cell growth and a block of 72% cells in G 0/1 phase of cycle are already obtained within only 3 days of treatment with the mixed ester of the invention. Instead the results obtained when the two active principles are separately added are those expected for the concentrations used. Indeed, BA at 0.2 mM concentration is totally ineffective in inducing an anti-proliferative effect or a cell cycle block, whereas retinoic acid at approximately 10 "5 M concentration (corresponding to a concentration of 0.1 mg/ml of the mixed ester) is cytotoxic, as it is well known.
• the mixed ester of the invention allows the administration of otherwise cytotoxic and therapeutically unsuitable doses of RA, and to obtain, at the same time, a pro-differentiating effect.
• the greater biological activity observed with the mixed esters compared to the association of the two mono-esters can be at least partially ascribed to the absence of a mechanistic antagonism between the two mono-esters in their binding kinetic to the same receptor.
• the applicants have observed that: - on a quantitative level, the mixed esters of HA with RA and BA, according to the present invention, show an anti-proliferative and cytotoxic or cytostatic effect that is dose-dependent and a pro-differentiating effect on cells derived from solid and systemic tumors with an activity significantly higher than that achieved by using the two individual acids, their physical mixture or their hyaluronic mono- esters; on a qualitative level each of butyric and retinoic acids, their physical mixture and the respective hyaluronic monoester all show a pro-differentiating effect which is significantly lower than the effect on cell growth inhibition. It should be noticed that both anti-proliferative and pro-differentiating effects are extremely useful in anti-tumor therapy and that only the esters of the present invention are able to produce significant effects on both parameters.
• the mixed esters with a degree of substitution comprised from 6 to 10 for both BA and RA, show an IC 50 (the concentration producing 50% anti-proliferative effect) lower than 1 nM.
• esters of the present invention is the controlled reduction of the molecular weight of HA. This feature reduces the steric hindrance of the final molecule (to the advantage for bioavailability), improves its solubility properties and reduces its viscosity in solution, making it more easily administrable to humans and animals. This is obtained without altering the site- specific delivering capacity of the native hyaluronic acid.
• the hyaluronic esters of the present invention have features of optimal solubility and low viscosity, making them suitable for easy formulations and easy and harmless administration even intravenously or intra-muscularly.
• the mixed esters of the invention can be prepared in the form of aqueous solution up to a concentration of at least 2 mg/ml.
• An additional embodiment of the present invention entails the process to synthesize the aforesaid mixed esters of HA. This process is characterized by an esterification step with retinoic acid derivatives performed prior to esterification with butyric acid derivatives.
• the process is characterized by the following steps, preferably in the order reported below : i) formation of an alcoholate of hyaluronic acid; ii) esterification of the alcoholate obtained in i) with retinoic acid derivatives to obtain a retinoic mono ester of hyaluronic acid; iii) esterification of the mono esters obtained in ii) with butyric acid derivatives to obtain the aforesaid mixed ester of hyaluronic acid.
• esterification with RA is carried out before the esterification with BA; moreover, it is even preferred that, prior to the esterification with RA, hyaluronic acid is transformed into the corresponding alcoholate according to the conditions indicated in point i).
• the HA that is used as starting material can be used as such or in a salt form; preferred example of hyaluronic acid salts are the quaternary ammonium salts, such as the tetrabutyl ammonium salt.
• Hyaluronic acid is commercially available and has a molecular weight generally ranging between 10 4 and 10 7 Daltons.
• the base employed in step i) for converting the hydroxyl groups of hyaluronic acid into their salts is preferably a quaternary ammonium hydroxide bearing C1-C5 alkyl radicals, such as tetrabutyl ammonium hydroxide.
• a C1-C5 alcohol preferably methanol, to dissolve hyaluronic acid.
• step ii) the alcoholate from step i) is preferably dispersed in dimethylformamide and added to a retinoyl-chloride solution that was separately prepared before.
• step iii) the following procedure is preferably followed: the retinoic monoester obtained in ii), preferably purified, is dispersed in dimethylformamide and triethylamine; butiryc anhydride and dimethylaminopyridine are then added to complete the esterification.
• the mixed ester can be further purified by washes, by dialysis, by passage through ionic exchange resins, etc.
• the present invention further comprises any pharmaceutical composition containing the above-said mixed esters, individually or in association with other compounds or with pharmaceutically acceptable excipients, which are more suitable for the formulation.
• examples of such pharmacological formulations are solutions, suspensions, soluble powders, tablets, granules, micro-capsules, soft or rigid capsules, coated tablets, suppositories, ovules, ointments, gel, etc.
• the invention further comprises the use in therapy of the aforesaid esters and of their pharmacological compositions.
• the invention comprises a therapeutic method to a patient in need of an antiproliferative and/or an antitumoral treatment, comprising administering to said subject a therapeutically effective amount of the mixed esters of the invention at therapeutically active doses .
• the present invention allows the use of the above specified mixed esters for the preparation of antitumoral medicaments with an anti-proliferative and a pro-differentiating action, particularly useful in the therapy of systemic tumors such as acute promyelocytic leukemia.
• the anti-proliferative properties of the esters of the invention make them useful for the treatment of all the diseases characterized by cellular hyper- proliferaton, as for example inflammatory bowel diseases, Crohn's disease, ulcerative colitis, psoriasis, hyperkeratosis, prostatic hyperplasia, synovial cell proliferation.
• the route of administration can be chosen, for example, among oral, intra-venous, intra-muscular, intra-peritoneal, rectal, intra-cavity, vaginal, trans-cutaneous, topic, etc.
• the present invention will now be further explained by way of the following examples, without being limited thereto. Experimental part Materials and methods. • Cell culture.
• MCF7, U937, HL-60 and HT29 cell lines were purchased from the American Tissue Culture Collection (Rockville, MS). MCF7 cells were grown in DMEM/F12 supplemented with 5% FBS, while U937 and HL-60 were grown in RPMI-1640 supplemented with 10% (v/v) foetal bovine serum. Cell proliferation.
• CD44, CD11a, CD11b, CD18 antigens was measured with specific murine monoclonal antibodies and secondary anti-mouse antibodies conjugated to FITC.
• FITC FITC-binding protein
• PI propidium iodide
• Rnase 10 kll/ml
• Nonidet P40 0.005%
• the fluorescence relative to PI was measured using a FACScan flow cytometer (Becton Dickinson) equipped with an argon laser at 488 nm excitation wavelength and a 610 nm filter.
• the fluorescence signal was collected in linear and logaritmic modes. Data were evaluated using the LYSIS II software.
• Example 1 Preparation of mixed ester with higher degree of substitution.
• several tests have been performed in order to obtain mixed butyric and retinoic esters of hyaluronic acid with different relative ratios of esterification with one or the other substituent. More specifically, we have chosen a sequential esterification approach where the first esterification step is with RA, because of its slower rate of reaction.
• the stoichiometric ratios of the reactants and also some operating conditions have been changed in order to obtain different degrees of esterification, although always the same general scheme of synthesis was maintained:
• the lyophilized HA-TBA alcoholate was dispersed in 300 ml of N.N-DMF and the retinoyl chloride solution, previously prepared, was then added drop-wise to this solution in approximately 30' and at RT.
• the reaction was incubated for additional 17 hours, always under magnetic stirring.
• the system obtained in this way was then divided in two identical aliquots: in the first aliquot (A) the retinoylation was stopped by concentration under vacuum to 1/3 of the initial volume of the solution and poured onto 3 volumes of ethyl ether. A precipitate was obtained that was re-suspended in 100 ml of N.N-DMF to which 300 ⁇ l of TEA are added.
• Both aliquots A and B are then treated with 640 ⁇ l of butyric anhydride and 2 g of DMAP, leaving the reaction stirring over-night sheltered from light and under nitrogen flow.
• the aliquots were filtered into a Gouch filter, washed with 3X100 ml of diethyl ether and purified by dialysis in 2 I of distilled water.
• the products purified in this way were then passed through a column of ion exchange resin in the sodium form and finally lyophilized. Yields and degrees of substitution of the two products HRE23 and HRE24 (synthetized from aliquot A and B) were respectively as follows: 0.8 g, d.s.
• HA-TBA 5.0 g of HA-TBA were solubilized in 20 ml of Methanol and 2.7 ml of a 40% (w/v) aqueous solution of TBA-OH, leaving the reaction for one night under magnetic stirring. The solvent was evaporated at reduced pressure and the product was lyophilized.
• HA-TBA 50 gr of HA-TBA were solubilized in 300 ml of methanol and treated with 27 ml of 40% TBA-OH solution.
• N.N-DMF and 5 ml of oxalyl chloride were added to 30 ml of diethyl-ether and a solution containing 12.5 g of retinoic acid in 100 ml of N,N-DMF was added.
• 2.5 I of alcoholate solution in N.N-DMF was added drop-wise to this solution during
• the selected experimental model consisted of a human mammary carcinoma cell line (MCF7) as example of solid tumor.
• MCF7 human mammary carcinoma cell line
• the effects induced by the mixed ester were compared with those obtained with the two active principles either in free form or bound to HA as monoesters.
• the presence of the CD44 receptor, specific for HA, was previously demonstrated (Coradini D, Pellizzaro C, Miglierini G, et al. Int J Cancer 81 :411-416, 1999).
• Figure 1 shows that, after 6 days of treatment, scalar doses of mixed esters (range 2-0.0001 mg/ml) exert higher anti-proliferative activity than corresponding concentrations of both mono-esters of butyric acid and retinoic acid (range 4- 0.0001 mg/ml).
• the promyelo-monocyitic HL-60 and U937 cell lines were chosen as examples of human systemic tumor. These cell lines undergo specific granulocytic (HL-60) or monocytic/macrophagic (U937) differentiation in presence of differentiating compounds such as retinoic acid.
• the mixed esters experimentally tested were characterized by the degrees of substitution specified in table 1. Table 1
• the selected experimental model consists of the U937 promyelo/monocytic cell line, in which the effect of mixed esters on a series of myelocyte/macrophage specific differentiation markers (CD11a, CD11b and CD18) has been studied.
• the resulting effect has been directly compared with that of the two active principles. Since the growth tests showed that the mixed ester induces maximal effect already at a concentration of 0.1 mg/ml, experiments have been carried out at this same concentration. The results have been compared with those obtained with butyric and retinoic acids at the same concentrations as in the mixed ester (respectively 0.2 mM and 10 5 M). Since the time of treatment was reduced to 3 days in these experiments, the effects on growth and cell cycle were also measured.
• Figure 6 indicates that, after 3 days of treatment, the mixed ester already induces the highest inhibition, with 72% of cells blocked in G0 /1 phase of the cell cycle.
• BA at 0.2 mM concentration was totally ineffective in inducing both an anti-proliferative effect and a cell cycle block
• RA at 10 "5 M concentration known to be cytotoxic and thus therapeutically unsuitable, significantly reduces cell growth with a concomitant cell block in the G 0/1 phase of the cycle.
• the cytotoxic effect of RA at this concentration was confirmed by alterations of cell morphology observed under the microscope.
• the effect of the mixed ester has been compared with the two mono-esters, separately or in association, that have been administered at concentrations corresponding to those in the mixed ester. It was observed that only the mixed ester was able at the same time to block cell growth (expressed as percentage of cells in the G0 /1 phase of the cycle) and to promote cell differentiation (expressed as percentage of cells expressing CD11 b antigen). In fact, as shown in Figure 7, the two mono-esters in association have a similar effect on the block of cell growth but were unable to induce any differentiation. This accounts for the remarkable potential of the mixed ester as compared to single active principles, their mono-esters and association of mono-esters. Example 6.
• Example 7 Evaluation of acute toxicity. with determination of LD50 and of sub- acute toxicity of the mixed esters in mouse.
• a mixed butyric-retinoic ester of hyaluronic acid was used, whose main physico-chemical characteristics are shown in table 3.
• Table 3 Physico-chemical characteristics of the mixed butyric-retinoic ester of hyaluronic acid.
• HBR hypothalamic hormone
• Example 8 Evaluation of the pharmacological activity of the mixed esters in mouse. Evaluation of the pharmacological activity of the mixed esters of the invention in the experimental model of TLX5 lymphoma was then performed with the same compound used for toxicological experiments, the characteristics of which are reported in table 3. As summarized in table 6, intraperitoneal treatment with HBR for 7 consecutive days significantly reduces the number of tumor cells present in the peritoneum, in animals sacrificed 8 days after implantation. Table 6. Effects of in vivo treatment with HBR on survival time and on tumor cell number in TLX5 lymphoma-bearing mice.
• mice were intraperitoneally injected with 100.000 TLX5 lymphoma cells. Twenty- four hours after tumor implantation, mice were then treated with HBR at a concentration of 6 or 12 ⁇ mol/mouse (200 ⁇ l or 400 ⁇ l intraperitoneum) for 7 consecutive days. The survival time was evaluated for 5 animals in each group (*) and the remaining animals were sacrificed at day7, in order to count peritoneal tumor cells ( ⁇ ).
• the TLX5 lymphoma was used as experimental model for screening different molecules and was normally used for evaluation of highly cytotoxic chemotherapeutic drugs exactly because of its extreme aggressiveness.
• a cell count reduction greater than 70% for drugs with anti-proliferative and/or pro-differentiating mechanisms of action, like HB and the HBR indicate a remarkable anti-tumor activity.
• drugs with anti-proliferative and/or pro-differentiating mechanisms of action like HB and the HBR

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